Method for reducing deoxynivalenol and its derivatives

Abstract

A method for reducing deoxynivalenol and its derivatives, and use of the method for reducing deoxynivalenol and / or its derivatives in grains, grain processing by-products, or grain products. The method reduces deoxynivalenol and / or its derivatives using low-temperature plasma utilizing high-pressure gas discharge. The method has the advantages of a high reduction rate, not requiring high temperatures, preserving the nutritional value and flavor of food, and leaving no residue.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to Chinese Patent Application No. 202010198993.3 (title: Method for reducing deoxynivalenol and its derivatives), filed on March 20, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of food science, and more particularly to a method for reducing deoxynivalenol and its derivatives. [Background technology]

[0003] Deoxynivalenol (DON), a deoxy derivative of nivalenol, is a water-soluble trichothecene mycotoxin produced by secondary metabolism in Fusarium species. Its chemical name is 3,7,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one and its molecular formula is C 15 H 20 O6, molecular weight 296.3. Deoxynivalenol, also known as vomitoxin, is widely distributed in nature in food crops such as corn, wheat, barley, maize, rye, and rice, and enters the food chain via food and its products (e.g., bread), resulting in serious contamination. 3-Acetyldeoxynivalenol (3-ADON) and 15-Acetyldeoxynivalenol (15-ADON) are the most common derivatives of DON and have recently been detected in grains and their products.

[0004] DON can cause a range of toxic reactions in humans and animals, including dizziness, vomiting, diarrhea, lethargy, decreased appetite, and central nervous system disorders. At the 56th meeting of the Joint FAO / WHO Expert Committee on Food Additives (JECFA), the provisional maximum tolerable daily intake (PMTDI) of DON was set at 1 μg / kg bw / d. At the 72nd meeting in 2010, JECFA further limited the intake of DON, 3-ADON, and 15-ADON compounds to 1 μg / kg bw / d. On October 27, 2017, the WHO International Agency for Research on Cancer published a preliminary classification of carcinogens, which included deoxynivalenol as a Category 3 carcinogen.

[0005] Conventional methods for reducing and controlling DON mainly include physical, chemical, and biological methods. However, these methods have drawbacks, such as energy consumption, a tendency to cause secondary pollution, and limitations on large-scale use. Effective methods for reducing 3-ADON and 15-ADON have yet to be found. Therefore, research into efficient, safe, and environmentally friendly DON reduction technologies is particularly important. Summary of the Invention

[0006] The present invention aims to overcome the current lack of an effective method for reducing the toxicity of deoxynivalenol (DON) derivatives (3-ADON, 15-ADON) by solving the problems of conventional techniques, such as complex procedures for reducing DON, the production of other contaminants, low reduction efficiency, and poor applicability. DON and its derivatives treated by the method of the present invention have low cytotoxicity, which can effectively ensure food safety.

[0007] In order to achieve the above object of the present invention, the following technical means are adopted. The present invention relates to a method for reducing deoxynivalenol and its derivatives by using low-temperature plasma utilizing high-pressure gas discharge.

[0008] Optionally, the derivative is an acetylated derivative of deoxynivalenol.

[0009] Optionally, the derivative is selected from 3-acetyldeoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON).

[0010] Optionally, the atmosphere of the high pressure gas discharge is one or more of air, oxygen-containing gas, nitrogen gas, and rare gas.

[0011] Optionally, the power supply output used for the high pressure gas discharge is a sinusoidal voltage.

[0012] Optionally, when reduction is performed, the voltage amplitude of the power supply is 3 to 8 kV and the frequency is 10 to 50 kHz.

[0013] Optionally, the duration of said reduction is ≧1 min.

[0014] Optionally, the reduction time is between 4 minutes and 6 minutes.

[0015] The present invention further relates to the use of said method in reducing deoxynivalenol and / or its derivatives in grains or grain products.

[0016] Optionally, the grain comprises: The grain may be any one selected from corn, wheat, barley, oat, rye, hulless barley, rice, zokubei, sorghum, Job's tears, Ethiopian teff, amaranth, quinoa seeds, kaniwa, buckwheat, and quinoa, or a combination thereof.

[0017] Compared with conventional technologies, the method of the present invention uses various components, such as ultraviolet rays, high-energy charged particles, and active substances (molecules, excited atoms, metastable atoms, and free radicals), generated during the low-temperature plasma generation process, which can interact comprehensively with DON and its derivatives to reduce toxins. Compared with conventional toxicity reduction technologies, the method of the present invention has the advantages of being highly effective, not requiring high temperatures, preserving the nutrition and flavor of food, and leaving no residue.

[0018] This invention uses low-temperature plasma treatment as its core technology, generating low-temperature plasma through high-pressure gas discharge, and conducting experimental research to reduce mycotoxins, thereby reducing DON concentrations and reducing the toxic effects of DON, providing a new approach and scientific basis for the construction of efficient, safe, and environmentally friendly DON reduction technology.

[0019] This method can reduce DON by more than 50%, and is highly effective against 3-ADON and 15-ADON, which are difficult to reduce by other methods. Furthermore, the reduction rate for 3-ADON can reach approximately 24% to 26%, and the reduction rate for 15-ADON can reach approximately 20%. [Brief explanation of the drawings]

[0020] In order to more clearly describe the technical solutions in the specific embodiments of the present invention, the following briefly describes the drawings that need to be used to describe the embodiments. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without creative efforts. [Figure 1] 1A is a schematic diagram of a plasma processing apparatus according to an embodiment of the present invention, and FIG. 1B and FIG. 1C are graphs showing the time-dependent changes in voltage and current, and the time-dependent changes in power and energy, respectively, during a plasma processing process according to an embodiment of the present invention. [Figure 2] This is a comparison of cell proliferation states before and after plasma treatment with DON (120 μg / ml) in one example of the present invention. [Figure 3]3A and 3B are cytotoxicity dose-effect curves of DON (120 μg / ml) before and after plasma treatment in one embodiment of the present invention. [Figure 4] 1 is a comparison of cell proliferation states before and after plasma treatment with different concentrations of 3-ADON in one embodiment of the present invention. [Figure 5] 5A and 5B are cytotoxicity dose-effect curves of 3-ADON before and after plasma treatment in one embodiment of the present invention. [Figure 6] 1 shows a comparison of cell proliferation states before and after plasma treatment with different concentrations of 15-ADON in one embodiment of the present invention. [Figure 7] 7A and 7B are cytotoxicity dose-effect curves of 15-ADON before and after plasma treatment in one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0021] Reference will now be made in detail to the embodiments of the invention, one or more examples of which are described below. The examples provided are for purposes of explanation, not limitation. Indeed, those skilled in the art may make various modifications and variations to the present invention without departing from the scope or spirit of the invention. For example, features illustrated or depicted as part of one embodiment may be applied to another embodiment to yield still further embodiments.

[0022] Therefore, it is intended that the present invention cover such modifications and variations as come within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present invention are disclosed in or will be obvious from the following detailed description. Those skilled in the art should understand that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the present invention.

[0023] The present invention relates to a method for reducing deoxynivalenol and its derivatives by using low-temperature plasma utilizing high-pressure gas discharge.

[0024] The present invention uses a high-voltage power supply connected to a plasma electrode to generate a micro-discharge. The various components generated during the low-temperature plasma generation process, such as ultraviolet rays, high-energy charged particles, and active substances (molecules, excited atoms, metastable atoms, and free radicals), can interact comprehensively with DON and its derivatives, thereby exerting a toxin-reducing effect. This technology offers significant advantages over conventional DON reduction techniques. Low-temperature plasma technology has the advantages of low cost, no waste, and no pollution, as well as high safety, no chemical residues, a short treatment process, and is environmentally friendly. The present invention can achieve ideal reduction rates for 3-ADON and 15-ADON, which are difficult to effectively reduce using physical, chemical, and biological reduction methods in conventional technologies.

[0025] From the above, the method provided by the present invention has good prospects for application.

[0026] In some embodiments, the derivative is an acetylated derivative of deoxynivalenol.

[0027] In some embodiments, the derivative is selected from 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol.

[0028] The gases used in the treatment process, in which a plasma is generated by a high-pressure gas discharge, can be, for example, oxygen-containing gases (e.g., O, H, O, NO, nitrogen gas, and air), inert gases (e.g., helium, neon, argon, krypton, xenon), or mixtures thereof. In some embodiments, the oxygen-containing gas is specifically O and air. The pressure can be atmospheric or lower.

[0029] In some embodiments, the reduction is carried out at room temperature (eg, 10°C to 40°C (eg, 18°C, 20°C, 25°C)).

[0030] In some embodiments, the power supply output used in the high pressure gas discharge is a high frequency AC voltage, and in some examples a sinusoidal voltage, although pulsed voltages and pulsed DC voltages may also be used.

[0031] In some embodiments, when reduction is performed, the voltage amplitude of the power supply is 3 to 8 kV and the frequency is 10 to 50 kHz.

[0032] In some embodiments, when reducing, the voltage amplitude of the power supply may be selected from 2, 5, 6 or 7 kV and the frequency may be selected from 15, 20, 25, 30, 35, 40 or 45 kHz, and in some embodiments is 25-35 kHz.

[0033] In some embodiments, a flat dielectric barrier discharge structure is used as the discharge electrode, circular aluminum plate electrodes are used as the upper and lower electrodes, the electrode diameter is 60 mm to 80 mm, the electrode edge radius is 8 mm to 12 mm, and the electrode effective area during discharge is 18 cm 2 ~21cm 2 (approx. 19.6cm 2 )

[0034] In some embodiments, a container for containing an object to be processed is placed on the lower electrode, and the container may be a glass petri dish.

[0035] In some embodiments, the reduction time is ≧1 min, e.g., 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min or more.

[0036] Although low-temperature plasma technology does not require harsh operating conditions such as high temperature, high pressure, or the addition of chemical reagents, it is generated under high-voltage discharge conditions, resulting in high power consumption and relatively high wastewater treatment costs. In the present invention, it was found that when reducing DON and its derivatives, increasing the treatment time did not significantly improve the effect when the reduction time was approximately 5 minutes. Therefore, in order to improve the energy utilization rate, reduce energy consumption, and reduce treatment costs, in one embodiment, the reduction time is set to 4 to 6 minutes, and in one example, it is approximately 5 minutes.

[0037] According to another aspect of the invention, the invention relates to the use of said method in reducing deoxynivalenol and / or its derivatives in grains, grain processing by-products or grain products.

[0038] In some embodiments, the grain comprises: The grain may be any one selected from corn, wheat, barley, oat, rye, hulless barley, rice, zokubei, sorghum, Job's tears, Ethiopian teff, amaranth, quinoa seeds, kaniwa, buckwheat, and quinoa, or a combination thereof.

[0039] The grain may be in the form of ground granules or powder.

[0040] Before reduction, the grain may be subjected to an impurity removal process, such as a vibrating screen, a de-stoner, or a self-circulating air separator.

[0041] In some embodiments, the grain product is a feed.

[0042] Hereinafter, the embodiments of the present invention will be described in detail with reference to examples.

[0043] Example The present invention verifies the DON reduction effect of low-temperature plasma through the following experiments.

[0044] method DON and its derivative solutions (DON, 3-ADON, 15-ADON) are treated with low-temperature plasma, and the reduction efficiency is detected by isotope dilution liquid chromatography-tandem mass spectrometry (LMS) on the treated solution. Cells are then cultured using the treated solution to measure the biological effects.

[0045] Materials and reagents Plasma generator; DON, 3-ADON, and 15-ADON samples; standards and isotope internal standards; petri dishes, centrifuge tubes, sterile water, cell culture reagents, and detection reagents such as CCK-8 kits.

[0046] Experimental procedure 1. Preparation of solutions 50 mg of DON per tube is dissolved in 5 ml of acetonitrile to give a concentration of 10 mg / ml, and then ultrapure water is added to dilute the solution to 0.12 mg / ml for use in plasma treatment. 10 mg / bottle of 3-ADON is dissolved in 1 ml of acetonitrile to give a concentration of 10 mg / ml, and then diluted to 0.12 mg / ml with ultrapure water for use in plasma treatment. 10 mg of 15-ADON per tube is dissolved in 1 ml of acetonitrile to give a concentration of 10 mg / ml, and then ultrapure water is added to dilute the solution to 0.12 mg / ml, and the solution is used for plasma treatment.

[0047] 2. Plasma treatment Figure 1A shows a schematic diagram of a plasma processing apparatus. The power supply used in this method is a high-voltage high-frequency power supply (HFHV20-1 type). This power supply consists of two parts: a control cabinet and a high-frequency transformer. The input is a 220V commercial frequency voltage, and the output is a sinusoidal voltage with an amplitude of 0 to 20kV (controllable) and a frequency of 10kHz to 60kHz (controllable). The voltage amplitude during the experiment was 3 to 8kV and the frequency was 30kHz. A flat dielectric barrier discharge structure was used as the discharge electrode, and circular aluminum plate electrodes were used as the upper and lower electrodes. The electrode diameter was 70mm, the electrode edge radius was 10mm, and the effective electrode area during discharge was approximately 19.6cm. 2A glass dish was placed on the lower electrode as a barrier medium, measuring 80 mm in diameter, 2 mm in thickness, and 2 mm in depth. After DON, 3-ADON, and 15-ADON were prepared, 7 ml of each solution was poured into the dish, generating plasma between the upper electrode and the solution surface, with an air gap of 2 mm. To compare the effects of low-temperature plasma treatment, treatment times were 1 minute (short time), 5 minutes (medium time), and 15 minutes (long time). Figure 1B shows graphs of the voltage and current over time during the plasma treatment process. Figure 1C shows graphs of the power and energy over time during the plasma treatment process.

[0048] 3. Verification of reduction rate According to the national standard GB5009.111, the concentrations of DON, 3-ADON, and 15-ADON after treatment were measured by isotope dilution liquid chromatography-tandem mass spectrometry to determine the reduction efficiency.

[0049] 4. Verification of biological effects Mouse embryonic fibroblast 3T3 cells were cultured as usual, and DON, 3-ADON, and 15-ADON mother solutions and plasma-treated solutions were used to prepare cell culture media. The test sample concentrations were DON (3200, 1600, 800, 400, 200, 100, 50, and 25 ng / ml), 3-ADON (12, 6, 3.2, 1.6, 0.8, 0.4, and 0.2 μg / ml), and 15-ADON (12, 6, 3.2, 1.6, 0.8, 0.4, and 0.2 μg / ml). A solvent control group and a normal culture medium control group were also included. The concentrations of the DON, 3-ADON, and 15-ADON plasma-treated solutions were adjusted according to the mother solution concentrations. After 24 hours of cell culture, cytotoxicity was tested using the CCK-8 assay to verify the biological effects.

[0050] result The results of the reduction of DON and its derivatives using low-temperature plasma are shown in the table. After 1 minute of treatment with low-temperature plasma, DON was decomposed by 16.3%, after 5 minutes of treatment, it was decomposed by approximately 52.1%, and after 15 minutes of treatment, no significant further reduction was observed. After 1, 5, and 15 minutes of treatment with low-temperature plasma, the reduction rate of 3-ADON was between 20 and 30%, and there was no tendency for the reduction rate to increase with increasing treatment time. The reduction results for 15-ADON were similar.

[0051] [Table 1]

[0052] [Table 2] JPEG0007774311000003.jpg161170

[0053] Results of biological effect verification tests The cytotoxicity results showed significant growth inhibition at 400 ng of DON, but the cytotoxic effect was not significant after plasma treatment (5 min) with the same concentration of DON. The cell growth status and dose-effect curve results are shown in Figure 2 and Figures 3A-3B, respectively.

[0054] The cytotoxicity results showed significant growth inhibition in the case of 3-ADON at 3.2 μg / ml, but the cytotoxic effect was not significant after plasma treatment (5 min) with the same concentration of 3-ADON. The cell proliferation status and dose-effect curve results are shown in Figure 4 and Figures 5A-5B, respectively.

[0055] The cytotoxicity results showed significant growth inhibition at 3.2 μg / ml of 15-ADON, but the cytotoxic effect was not significant after plasma treatment (5 min) with the same concentration of 15-ADON. The cell proliferation status and dose-effect curve results are shown in Figure 6 and Figures 7A-7B, respectively.

[0056] Although the technical features of the above-described embodiments can be combined in any manner, for the sake of convenience, not all possible combinations of the technical features of the above-described embodiments are described. However, it should be understood that as long as there is no contradiction in the combination of these features, it is included in the scope of the description of this specification.

[0057] The above examples merely illustrate some embodiments of the present invention, and although the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. Those skilled in the art may make some modifications and improvements without departing from the spirit of the present invention, and all such modifications and improvements are within the scope of the present invention. Therefore, the scope of the present invention is defined by the appended claims.

[0058] References 1.Samr M,Resnik SL,Gonzfilez HHL,et al.Deoxynivalenol reduction during the frying process of turnover pie covers[J].Food Control,2007,18(10):1295-1299. 2.Wu L,Wang BJ.Evaluation on levels and conversion profiles of DON,3-ADON,and 15-ADON during bread making process.Food Chemistry,2015,185:509-516 3. Sabater-Vilar M, Malekinejad H, Selman MHJ, et al. In vitro assessment of adsorbents aiming to prevent deoxynivalenol and zearalenone mycotoxicoses[J]. Mycopathologia, 2007, 163(2):81-90. 4. Zhang Kun, Bian Ke, Guan Erqi, Zhang Xinsheng, Xu Mengmeng. Study on the Decomposition of Deoxynivalenol by Electron Beam Irradiation. Food and Feed Industry, 2014, 4:13-16 5. State Administration for Quality Supervision, Inspection and Quarantine of the People's Republic of China <Industry Standard for Import and Export Quarantine of the People's Republic of China: Good Radiation Guidelines for Imported and Exported Radioactive Foods SN / T 1887-2007> 6. Ministry of Health of the People's Republic of China <National Food Safety Standard for Prepackaged Foods GB7718-2011> 7. Xie Maochang, Wang Mingzu. Chemical method for removal of toxin (DON) [J]. Journal of Shanghai Agricultural Sciences, 2000, 16(1): 58-61. 8.Niderkorn V, Boudra H, Morgavi DP.Binding of Fusarium mycotoxins by fermentative bacteria in vitro.Journal of Applied Microbiology.2006,101:849-856 9.WANG Gang,WANG Yanxia,JI Fang,et al.Biodegradation of deoxynivalenol and its derivatives by Devosia insulae A16[J].Food Chemistry,2019,276:436-442.

Claims

1. A method for reducing deoxynivalenol derivatives contained in a treatment target, comprising: Deoxynivalenol derivatives are reduced by low-temperature plasma using high-pressure gas discharge. the derivative is selected from 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol; the high-pressure gas discharge atmosphere is air, The output of the power supply used for the high-pressure gas discharge is a sine wave voltage, and when reduction is performed, the voltage amplitude of the power supply is 3-8 kV, the frequency is 10-50 kHz, and the reduction time is ≧1 min; A flat dielectric barrier discharge structure is used as the discharge electrode, and circular aluminum plate electrodes are used as the upper and lower electrodes. The electrode diameter is 60 mm to 80 mm, the electrode edge radius is 8 mm to 12 mm, and the effective electrode area during discharge is 18 cm ２ ~21cm ２ and A method characterized in that a container for containing the object to be treated is placed on a lower electrode, the object to be treated is contained in the container, and low-temperature plasma is generated between the upper electrode and the object to be treated.

2. 2. The method of claim 1, wherein the reduction time is between 4 minutes and 6 minutes.

3. Use of the method described in any one of claims 1 to 2 in reducing deoxynivalenol derivatives in grains, grain processing by-products or grain products as the object to be treated.

4. 4. The use according to claim 3, wherein the cereal is any one or combination of cereals selected from corn, wheat, barley, oat, rye, hulless barley, rice, zokubei, sorghum, Job's tears, Ethiopian teff, amaranth, quinoa seeds, kaniwa, buckwheat, and quinoa.

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